Abstract
We have reported that the major endogenous estrogen, 17β-estradiol (E2), protects against oxidative injury during ethanol withdrawal (EW) in a cultured hippocampal cell line (HT22). Here, we investigated whether the pro-oxidant nature of EW mediates opening of the mitochondrial membrane permeability transition pore (PTP) in a manner protected by E2. Excess PTP opening provokes mitochondrial membrane swelling (MMS) and the collapse of membrane potential (ΔΨm). HT22 cells were collected at the end of ethanol exposure (100 mM) for 24 h or at 4 h of EW to assess MMS by monitoring absorbance decline at 540 nm and to assess ΔΨm using flow cytometry. Protective effects of E2 on PTP were compared with an antioxidant butylated hydroxytoluene (BHT) and an E2 analog, ZYC26 [(3-hydroxy-2-adamantyl(1)-4-methyl-estra-1,3,5(10)-17-one], with higher antioxidant potency than E2. To assess cellular consequences of PTP opening, effects of a PTP inhibitor (cyclosporin A) on EW-induced cell death were assessed using the calcein assay. Major findings were that: 1) EW resulted in rapid MMS and ΔΨm collapse; 2) cyclosporin A attenuated EW-induced cell death; and 3) E2 treatment restricted to the EW phase protected against the PTP opening more prominently than BHT and to a similar degree to ZYC26. These findings suggest that EW provokes PTP opening partly but not entirely through the pro-oxidant nature and that E2 counteracts EW-associated factors to protect against the PTP opening.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants AA013864, AA015982].
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doi:10.1124/jpet.108.146829.
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ABBREVIATIONS: EW, ethanol withdrawal; E2, 17β-estradiol; ROS, reactive oxygen species; PTP, mitochondrial membrane permeability transition pore; BHT, butylated hydroxytoluene; ZYC26, (3-hydroxy-2-adamantyl(1)-4-methyl-estra-1,3,5(10)-17-one; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; MMS, mitochondrial membrane swelling; AM, acetoxymethyl ester; ANOVA, analysis of variance.
- Received October 27, 2008.
- Accepted December 1, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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